Continuous monitoring of penicillin G and gentamicin in allantoic fluid of pregnant pony mares by in vivo microdialysis

REASONS FOR PERFORMING STUDY: Most current treatments for placentitis in mares are empirical with few control studies to evaluate their effectiveness. OBJECTIVE: To monitor drug concentrations in allantoic fluid of pregnant pony mares using in vivo microdialysis and establish if this method would be useful for determining allantoic concentrations of drugs in normal mares and those with placentitis. METHODS: Five late gestational pony mares had microdialysis probes inserted into the allantoic fluid using transabdominal ultrasound-guided allantocentesis. Single injections of penicillin G (22,000 u/kg), gentamicin (6.6 mg/kg bwt) and flunixin meglumine (1 mg/kg bwt) were administered i.v. and dialysate samples collected continuously for 24 h. In a separate study, drug concentrations were monitored in allantoic fluid of 2 mares with experimental placentitis induced by intracervical inoculation with Streptococcus equi ssp. zooepidemicus. Drug concentrations were measured by high performance liquid chromatography (penicillin G, flunixin meglumine) or enzyme-linked immunosorbent assay (gentamicin). RESULTS: Penicillin G and gentamicin achieved average peak concentrations of 9.8+/-2.2 and 8.5+/-3.1 microg/ml, respectively, in allantoic fluid of noninfected mares. Pharmacokinetic comparisons indicate that penicillin G persists much longer in allantoic fluid than blood, whereas gentamicin exhibited similar profiles in the 2 compartments. Flunixin meglumine was not detected in allantoic fluid. In infected mares, penicillin G achieved a similar peak concentration in allantoic fluid (11.2 microg/ml) whereas peak gentamicin concentration (3.9 microg/ml) appeared to be reduced relative to drug concentrations in noninfected mares. CONCLUSIONS: Microdialysis is a useful technique for continuous in vivo monitoring of drugs in equine allantoic fluid. Our results indicate that penicillin G and gentamicin undergo effective placental transfer in pregnant mares and in 2 mares that transplacental drug transfer may be altered selectively if active placental infection is present. POTENTIAL RELEVANCE: Further studies are needed to evaluate the feasibility of using increased dose intervals for penicillin G and an increased dose rate of gentamicin to effectively combat placental infections in mares.     

Quantification of Mannheimia haemolytica leukotoxin by indirect ELISA

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.     

Phenotypic and genetic characterization of enteropathogenic Escherichia coli (EPEC) and entero-aggregative E. coli (EAEC) from diarrhoeal and non-diarrhoeal children in Libya

A total of 50 Escherichia coli strains isolated in a Libyan hospital (20 from children with diarrhoea and 30 from healthy children) were investigated for their pathotypes and virulence traits. Altogether nine eae-positive (enteropathogenic E. coli, EPEC) and nine aggR-positive (entero-aggregative E. coli, EAEC) strains were identified. Significantly (P=0.001) more EPEC strains were identified from diarrhoeal patients (n=8) than from healthy controls (n=1), while six EAEC strains were identified from diarrhoeal and three from healthy children. Typical (eae(+), EAF(+), bfp(+)) EPEC strains (n=6) belonged to classical EPEC serogroups O55, O114, O127 and showed localized adherence on Hela cells. EAEC strains revealed genetic heterogeneity but uniformly adhered to HeLa cultures in an entero-aggregative adherence pattern. Antibiotic resistance frequently, characterized the strains. Sixty-eight percentage of the strains were resistant against at least one antibiotic and 30% harbored a class 1 integron independently of their clinical background. This is the first report from North Africa demonstrating the significance of EPEC and EAEC.     

Quantification of surgical blood loss

OBJECTIVE: To compare gravimetric and colorimetric methods of quantifying surgical blood loss, and to determine if there is a correlation between preoperative hemostatic tests (buccal mucosa bleeding time [BMBT] and intraoperative blood loss). STUDY DESIGN: Prospective clinical study. ANIMALS: Dogs (n=15) admitted for cutaneous tumor excision, orthopedic procedure, or exploratory laparotomy. METHODS: Intraoperative blood loss was quantified by measuring irrigation fluid and weighing surgical sponges used for blood and fluid collection during surgery. Results of gravimetric measurements were then correlated to blood loss quantified using spectrophotometric analysis of hemoglobin (Hb) content. Hemostatic variables including BMBT were measured before surgery and compared with the calculated amount of blood loss. RESULTS: Blood loss quantified by gravimetric measurement showed a significant correlation with colorimetric determination of Hb content in surgical sponges and collected irrigation fluid (r=0.93, P<.0001). BMBT correlated weakly but significantly with intraoperative blood loss (r=0.56, P<.05). CONCLUSIONS: Quantifying intraoperative blood loss using spectrophotometric Hb analysis accurately assessed the amount of blood loss; however, it is a time-consuming procedure, primarily applicable as a research tool. Gravimetric evaluation of intraoperative blood loss was found to be an accurate method, which can be recommended for use in a clinical setting. CLINICAL RELEVANCE: Estimation of blood loss using a gravimetric method is accurate and applicable in the clinical setting and provides surgeons with a simple and objective tool to evaluate intraoperative blood loss.     

Application of polymerase chain reaction and virus isolation techniques for the detection of viruses in aborted and newborn foals

The occurrence of two important pathogens, equine herpesvirus 1 (EHV1) and equine arteritis virus (EAV) causing abortions, perinatal foal mortality and respiratory disease, was investigated by polymerase chain reaction (PCR) and virus isolation to demonstrate the presence of abortigenic viruses in samples from 248 horse fetuses in Hungary. We found 26 EHV1- and 4 EAV-positive aborted or prematurely born foals from 16 and 4 outbreaks, respectively, proving that despite the widely applied vaccination, EHV1 is a far more important cause of abortions in the studs than EAV. We compared the virus content of different organs of the fetuses by PCR and isolation to identify the organ most suitable for virus demonstration. Our investigations indicate that the quantity of both viruses is highest in the lungs; therefore, according to our observations, in positive cases the probability of detection is highest from lung samples of aborted or newborn foals. Both the PCR and the virus isolation results revealed that the liver, though widely used, is not the best organ to sample either for EHV1 or for EAV detection. From the analysis of the epidemiological data, we tried to estimate the importance of the two viruses in the Hungarian horse population.     

Detection of pigeon circovirus in cloacal swabs: implications for diagnosis, epidemiology and control

Pigeon circovirus (picv) was detected in cloacal swab samples by means of a newly-developed, sensitive pcr. An initial investigation of 17 Belgian racing pigeons aged up to eight months showed that rates of detection of 88 per cent and above were achieved using samples of cloacal swab, blood and bursa of Fabricius. The sampling of 15 caged pigeons six times when they were from three to 31 weeks of age indicated that picv infections were more readily detected in cloacal swabs than in blood, and that the virus could be detected in cloacal swabs for longer periods after infection than in blood. picv infections were detected in cloacal swabs from 38 of 47 young pigeons aged from two to 31 weeks, from 12 racing lofts, which had clinical signs including diarrhoea and weight loss, regurgitation and respiratory signs. Samples from birds from two infected lofts indicated that picv could be detected in some birds for at least 27 weeks. Although nine of 14 pigeons aged from 32 to 45 weeks were virus-positive, picv was detected in only one of 18 adult pigeons that originated from four infected lofts.     

Genotyping of Toll-like receptor 4, myeloid differentiation factor 2 and CD-14 in the horse: an investigation into the influence of genetic polymorphisms on the LPS induced TNF-alpha response in equine whole blood

The inter- and intra-species differences in the response to lipopolysaccharides (LPS) are well recognised in mammalian species. It has been hypothesized that these differences can be attributed to genetic polymorphisms in the components involved in LPS signal transduction. These components include the cluster of differentiation factor 14 (CD-14), a membrane bound protein on the surface of mononuclear cells that recognises LPS and a receptor complex consisting of Toll-like receptor-4 (TLR-4) and myeloid differentiation factor-2 (MD-2). Sequencing of these three proteins in humans and mice revealed that all three are susceptible to polymorphic alterations, influencing the response to LPS. Previous experiments in the horse showed large inter-individual variations in the response to LPS. With the aim to assess this inter-individual variation, we performed a whole blood assay in 10 healthy horses as a functional assay to study the responsiveness to LPS. In 3 out of the 10 horses, LPS-induced TNF-alpha production was significantly lower compared to the overall mean. Subsequently the entire cDNA sequence encoding for the TLR-4, MD-2 and CD-14 protein was documented for each horse. Although mutations were observed in the sequence of TLR-4, these could not be related to an altered response to LPS in the concentration used in this study, as determined in the whole blood assay. Despite the various mutations found in the TLR-4 receptor protein, no alterations could be found in either the MD-2 or CD-14 gene, which are obviously more conserved structures.     

Canine limbal melanoma: 30 cases (1992-2004). Part 1. Signalment, clinical and histological features and pedigree analysis

OBJECTIVES: (1) To review the signalment, clinical, and histological features of canine limbal melanoma; (2) to perform pedigree analysis on breeds predisposed to limbal melanoma to establish if common ancestry exists; and (3) to investigate if any ancestral relationship exists between canine limbal melanoma and canine anterior uveal melanoma (CAUM). DESIGN: Retrospective study. ANIMALS STUDIED: Thirty dogs with limbal melanoma. METHODS: Medical records of patients were reviewed. Follow-up information was obtained by re-examination of patients or telecommunications with the referring veterinary surgeons or the owners. Pedigrees were analyzed for common ancestry amongst affected dogs. RESULTS The mean age (+/- SD) at diagnosis was 6.2 (+/- 2.75) years with a range from 1 to 11 years. There was a bimodal distribution of ages with a peak at 3-4 years and a peak at 7-10 years. There was no eye predilection or predisposition for sex or coat color. Twenty-five (83%) of the limbal melanomas occurred within a dorsal arc from the dorsomedial to the ventrolateral limbus. Golden retrievers were four times more common in the melanoma group compared to the Animal Health Trust population (P < 0.0001). Labrador retrievers were three times more common in the melanoma group (P=0.01). Pedigree analysis on eight Golden retrievers [limbal melanoma (n=5), CAUM (n=2) and diffuse ocular melanosis (n=1)], revealed a pattern of inter-relatedness consistent with the condition(s) being caused, at least in part, by a genetic mutation(s). A similar level of inter relatedness was evident in six Labrador retrievers (limbal melanoma (n=2) and CAUM (n=4)). In 5/22 cases (23%), histological features suggestive of malignancy were present including intratumor necrosis in 4/22 cases (18%) and cellular atypia in 1/22 cases (5%). CONCLUSIONS: In Golden and Labrador retrievers there is evidence that limbal melanomas, CAUM and ocular melanosis are at least in part heritable and that the same genetic mutation(s) may be causally associated with melanocytic disease at different ocular sites. The same genetic mutation(s) may be present in these two breeds. Histology should be performed on all cases to identify those with greater malignant potential.     

Drug Efficacy of Terbinafine Hydrochloride (Lamisil®) During Oral Treatment of Cats,Experimentally Infected with Microsporum canis

Cats represent a primary source of Microsporum canis infections in humans. Terbinafine hydrochloride (Lamisil^®) is commonly used in the treatment of microsporosis in humans as its fungicidal action permits short periods of treatment. The aim of the present study was to estimate the efficacy of the drug in cats. Nine cats were experimentally infected with M. canis and treated with terbinafine hydrochloride at a dose of 10–20 mg/kg (once daily, SID; low‐dose group, LDG). Another nine cats were similarly infected and treated with 30–40 mg/kg SID (high‐dose group, HDG) and a further nine cats were also infected and left untreated (control group, CG). The general condition of the cats was observed daily and their clinical symptoms evaluated weekly. The cats recovery was monitored using the Wood's lamp illumination test and microscopic and fungal culture examinations. The general condition of the cats during the study was good. The cure rates of the LDG were not significantly different from the CG at any period during the treatment. However, the HDG cure rates differed significantly from the other two groups. After 109 days of treatment, when all nine cats of the HDG were healed, seven cats of the LDG and all the cats in the CG were still M. canis‐positive. This study shows that dosages of 10–20 mg/kg SID of terbinafine hydrochloride are not sufficient to terminate an experimental M. canis infection in cats within an acceptable period of time. Terbinafine hydrochloride can be used to treat dermatophytosis in cats, but a higher dosage, 30–40 mg/kg SID, should be used to achieve a cure.